diabetes research and clinical practice 84 (2009) 123–131
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Diabetes Research and Clinical Practice journal homepage: www.elsevier.com/locate/diabres
Glucose Tranporter-4 expression in monocytes: A systematic review Yorgi Mavros a,*, David Simar b, Maria A. Fiatarone Singh a,c,d a
Exercise, Health and Performance Faculty Research Group, Faculty of Health Sciences, University of Sydney, East Street, Lidcombe, Sydney, NSW 2141, Australia b School of Medical Sciences, University of New South Wales, Sydney, NSW, Australia c Faculty of Medicine, University of Syndey, Sydney, NSW, Australia d Hebrew SeniorLife and Jean Mayer USDA Human Nutrition Center on Aging, Tufts University, Boston, MA, USA
article info
abstract
Article history:
Objective: The purpose of this review was to systematically assess the extent of current
Received 17 June 2008
knowledge of Glucose Tranporter-4 (GLUT-4) expression in monocytes in humans to address
Received in revised form
its potential use as a non-invasive and reliable model to investigate the relationships
10 December 2008
between insulin signalling, GLUT-4 expression and insulin action in vivo.
Accepted 9 February 2009
Method: Electronic database searches were performed with the keywords ‘monocyte’,
Published on line 14 March 2009
‘leukocyte’ and ‘white blood cells’, and the terms ‘GLUT’, ‘glucose transporter’ and ‘SLC2A4’ (solute carrier family 2 member 4). Studies were examined for robustness of design and
Keywords:
outcomes by consensus of three reviewers.
Monocyte
Results: Six cross-sectional or observational studies met the criteria for review. Insulin-
GLUT-4
stimulated GLUT-4 expression in monocytes from subjects likely to have impaired insulin
Diabetes
sensitivity appeared blunted relative to healthy subjects.
Insulin resistance
Conclusion: The available results provide evidence that monocyte GLUT-4 translocation does
Flow cytometry
occur in response to acute insulin exposure, and may be sensitive to the relative state of insulin resistance of the individual. However, due to the limited quantity and robustness of published data, the ultimate utility of monocyte GLUT-4 expression as an index of whole body insulin responsiveness and the clinical relevance of this methodology is unresolved at this time. Crown Copyright # 2009 Published by Elsevier Ireland Ltd. All rights reserved.
1.
Introduction
1.1.
Glucose transporters
Of particular interest to the study of the pathophysiology of insulin resistance and type 2 diabetes is the Glucose Tranporter-4 (GLUT-4), which is an isoform of the GLUT family found primarily in insulin sensitive tissues [1]. In fasting and non-exercise (resting) conditions, GLUT-4 is located in small intracellular tubulo-vesicular organelles
within skeletal muscle and other insulin sensitive cells. In response to insulin stimulation, it translocates to the plasma membrane of the cell [2–5]. Studies of the kinetics of GLUT-4 translocation have shown that it has a high affinity for glucose, with a Km between 2 and 5 mM [6]. As a result, GLUT-4 is constantly operating at close to its Vmax over physiological ranges for blood glucose, and so the rate of glucose transport into cells is not determined by glucose concentrations per se, but rather by the response of GLUT-4 to insulin signalling pathways. Secondly, stimulation by insulin increases the Vmax
* Corresponding author. Tel.: +61 2 9351 9279; fax: +61 2 9351 9204. E-mail address:
[email protected] (Y. Mavros). 0168-8227/$ – see front matter . Crown Copyright # 2009 Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2009.02.014
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diabetes research and clinical practice 84 (2009) 123–131
of glucose transport into cells without affecting the Km of the GLUT-4 transporter [7], suggesting that the action of insulin does not cause an intrinsic change in the structure of the GLUT-4 protein, but rather an alteration in the number of GLUT-4 transporters at the membrane of the cells.
1.2.
Insulin and GLUT-4
When insulin binds to the extracellular subunit of its receptor, there is an activation of its intrinsic tyrosine kinase, which then phosphorylates and activates intracellular receptor substrates, including members of the insulin substrate family (IRS). Once activated, these IRS proteins activate phospatidylinositol 3-kinase (PI 3-kinase), resulting in GLUT-4 translocation to the membrane [8].
1.3. Insulin resistance and GLUT-4 in type 2 diabetes mellitus Although not all studies are in agreement [6], most support exists for a reduction in insulin stimulated GLUT-4 translocation rather than an altered total GLUT-4 content in the skeletal muscle of subjects with type 2 diabetes [2]. This suggests that it is an impairment in insulin-stimulated GLUT-4 translocation that results in the reduction of peripheral glucose uptake within the insulin resistant population [9]. Interestingly, the altered translocation of GLUT-4 in response to insulin seen in the insulin resistant population is not evident during contraction of skeletal muscle. An in vivo study of obese subjects with type 2 diabetes found a normal increase in whole body glucose disposal in response to an acute bout of moderate-intensity aerobic exercise [10]. It is possible that the pathways for GLUT-4 translocation secondary to insulin stimulation and muscle contraction are independent of each other [13].
1.4.
Measurement of GLUT-4
The expression of GLUT-4 on the plasma membrane of peripheral tissues (namely skeletal muscle) is commonly measured through a muscle biopsy, as this gives a direct measure of its expression in the tissue responsible for the majority of glucose disposal. However, criticism of the use of muscle biopsies has been put forward, given the variability which may arise from measurements taken in only a small fraction of the whole muscle, as well as the potential error due to extrapolating results from one muscle to another [11]. It has also been shown that GLUT-4 expression within the vastus lateralis muscle is fibre type-dependent, with slow twitch fibres (Type I) showing a greater density of GLUT-4 than fast twitch fibres (Type II) [12]. In addition, insulin-mediated glucose uptake is positively correlated with the proportion of Type I fibers in an individual [13]. The observation of an insulin receptor on monocytes contributed to the development of in vitro studies examining the effects of insulin binding to these cells in human blood [14]. Such studies have shown that insulin has an immediate stimulating effect on the uptake of radioactively labeled glucose into monocytes [15]. Physiological concentrations of insulin were found to increase monocyte glucose uptake and
lactate release in healthy subjects [16], as well as to increase monocyte glucose uptake in subjects with type 1 diabetes [17]. Recently, GLUT-4 levels on the plasma membrane of monocytes have been measured under resting and stimulated conditions both in vivo and in vitro. If this technique proves to be a reliable index of insulin sensitivity and glucose transport at the whole body level, it would markedly facilitate investigation in this field, given the feasibility of peripheral blood sampling relative to the standard muscle biopsy technique. The ability to monitor response to pharmacological, dietary, and exercise interventions in those with insulin resistance and type 2 diabetes via minimally-invasive peripheral blood sampling would have obvious and extensive clinical utility. Due to the paucity of information concerning the use of this technique to assess insulin resistance, the purpose of this review was to examine all published literature on monocyte GLUT-4 levels, to assess the extent of current knowledge in this area, including the reported validity and precision of this method, adaptations to altered metabolic conditions and physiologic stimuli, and to identify directions for future research.
2.
Research methods and procedure
A systematic review of the literature, regardless of study design, investigating GLUT-4 expression at the plasma membrane of monocytes using flow cytometry was conducted. Alternative methods to flow cytometry such as immunoblotting were excluded due to their insensitivity in detecting GLUT-4 expression in monocytes [17,18].
2.1.
Literature search
Computerised database searches were performed using MEDLINE (1950 to September 2007), CINAHL (1982 to present), SPORTS DISCUS (1830 to May 2007), EMBASE (1966–2007) and PRE-MEDLINE on the 17th of September 2007 and were updated in March 2008. The search terms ‘monocyte’, ‘leukocyte’ and ‘white blood cells’ were combined using ‘OR’, and the terms ‘GLUT’, ‘glucose transporter’ and SLC2A4 (solute carrier family 2 member 4) were combined using ‘OR’. Results from these two searches were then combined using ‘AND’. All searches were performed using ‘all fields’. Hand searching of retrieved articles and related citation indexes was also performed, as well as an author search of all retrieved articles.
2.2.
Inclusion/exclusion criteria
Studies were included if GLUT-4 expression on the membrane of monocytes using flow cytometry was an outcome of the paper. Studies were not restricted by study design, subject characteristics or intervention due to the novelty of the method and paucity of studies. Other inclusion criteria included: published, full length articles, and English language. A single author located and extracted data, while two other authors reviewed all retrieved articles for inclusion and accuracy of data extraction. Any disputes were resolved by consensus.
diabetes research and clinical practice 84 (2009) 123–131
3.
Results
3.1.
Studies retrieved
A summary of the search results is provided in Fig. 1. The literature search resulted in 301 studies. Literature titles were examined, excluding 291 studies. The remaining 10 abstracts were examined manually, and 4 studies were excluded. The whole text of the six remaining studies were examined and found to meet the criteria for review [19–24].
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incubating the sample with either wortmannin or IRA prior to incubation with insulin blocked GLUT-4 translocation to the monocyte membrane, confirming the role of insulin as a primary mediator of this process [19–21].
3.8.
Basal GLUT-4 expression in monocytes
All the studies included in the review were observational studies of either a single cohort [19] or cross-sectional comparisons of cohorts potentially differing in insulin sensitivity [19–24]. All studies were performed with subjects in a resting, fasted state. No study has reported post-prandial monocyte GLUT 4 levels, or monocyte GLUT 4 expression following an oral glucose challenge, meal tolerance test, exercise or pharmacological exposure.
Under basal conditions, GLUT-4 expression on monocytes was found to be higher in hyperthyroid subjects when compared to euthyroid subjects (data not provided) [20,24]. It was also reported to be lower in women (without gestational diabetes) during the second trimester of pregnancy and to be higher at term when compared to non-pregnant controls [22]. Hypoglycaemia (of unspecified duration or aetiology), however, was unrelated to GLUT-4 expression in healthy subjects [23]. Finally, no correlations were found between basal monocyte GLUT-4 expression in healthy subjects and their BMI, fasting plasma insulin or insulin resistance as assessed by HOMA2-IR (homeostatic model assessment) [21], although it must be noted that the sample size used to test these relationshps was very small (n = 6), suggesting the possibility of type II error.
3.3.
3.9.
3.2.
Study design
Cohort characteristics
A summary of the cohort characteristics is provided in Table 1. A total of 152 subjects were enrolled across 6 trials, which ranged in size from 12 (two studies) to 38 subjects. Only two of the studies provided a gender breakdown [22,23], both of which involved only women.
3.4.
Age
Age data was reported in four of the studies [19–21,24]. These studies reported mean ages of 40 2 [19], 35 2 and 40 4 [20], 30 2 and 34 3 [21] years, 37 2 and 41 2 [24] with a pooled mean age of 37 year across these 105 subjects. No data on older adults or children are available to our knowledge.
3.5.
Health status
The health status of subjects studied included those with overweight/obesity [21,24], pregnancy [22], hyperthyroidism (untreated) [20,24], and hypoglycemia (etiology unkown) [23]. No subjects with type 1 or type 2 diabetes have been investigated using this technique to date.
3.6.
Details of methodology
Table 2 displays a summary of the monocyte GLUT-4 methodology and results in all studies.
3.7.
Precision and validation of the technique
In two studies, validation of the specificity of GLUT-4 antisera used for monocyte staining was performed [19,21]. Three studies provided data on construct (face) validity, by confirming the specificity of the GLUT-4 translocation to insulin stimulation by preparing separate samples incubated with wortmannin [20,21], while one study used an anti-insulin receptor antibody (IRA) [19]. These three studies showed that
GLUT-4 expression after monocyte stimulation
Exposure of monocytes to either LPS [21], insulin [19–21,24], IGF-1 [24], insulin and LPS [21] or insulin and IGF-1 [24], has been shown to increase monocyte GLUT-4 expression, with differences in the magnitude of the response seen generally consistent with the clinical state of the subjects under study. In these four studies, incubation of monocytes from healthy subjects with insulin was found to increase the translocation of GLUT-4 by 50% ( p < 0.001) [19], 54% ( p < 0.001) [20], 24% ( p = 0.003) [19,21] and 39% ( p < 0.005) [24]. One study reported that the ED50 (median effective dose) value of insulin stimulation was within the physiological range of insulin levels at 20 mU/L [19]. Two studies reported that a maximal effect was achieved with an insulin concentration of 100 mU/ L, a level associated with clinical insulin resistance [19,21]. Activation of monocytes with lipopolysaccharide (LPS) was also found to increase GLUT-4 translocation ( p = 0.0004, data not provided) in the absence of insulin [21]. Subsequent exposure to increasing insulin concentration from 0 to 100 mU/L was found to increase GLUT-4 translocation by an additional 24% ( p = 0.006) in LPS-activated monocytes. Two studies compared responsiveness to insulin in hyperthyroid cohorts compared to healthy subjects [20,24]. In hyperthyroid subjects, monocytes were found to have a lower relative rate of responsiveness to insulin, with an increase in GLUT-4 of 25% and 10% observed in hyperthyroid subjects compared to 54% and 39% observed in healthy subjects [20,24]. However, absolute levels of GLUT-4 tended to be higher at all insulin concentration in hyperthyroid subjects [20,24], with significant differences at 0 and 10 mU/L ( p < 0.05) [20]. Stimulation of monocytes with IGF-1 increased GLUT-4 translocation by 21% in euthyroid subjects ( p < 0.005), and 10% in hyperthyroid subjects ( p = 0.003) [24]. Combined exposure of monocytes to IGF-1 and insulin in a healthy population did not augment the increase in GLUT-4 translocation statistically as compared to exposure to each hormone individually (data not provided).
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diabetes research and clinical practice 84 (2009) 123–131
Fig. 1 – Summary of search results.
In obese/insulin resistant subjects (HOMA-IR: 3.3 0.6), incubation with 100 mU/L of insulin did not cause a significant increase in GLUT-4 translocation ( p = 0.2) in contrast to the significant rise observed in healthy subjects [20].
3.10. Metabolic effects of GLUT-4 translocation in monocytes Two studies have shown that increases in insulin concentration correspond to increases in GLUT-4 translocation as well as
diabetes research and clinical practice 84 (2009) 123–131
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Table 1 – Subject characteristics. Citation
Study design
Sample size (n)
Age
Gender
BMI (kg/m2) Health status
Korgun et al. [22]
Cross-sectional
12 Non-pregnant women 91st trimester 72nd trimester 73rd trimester (term)
NR
Females
